The present invention relates to a method of immunologically measuring human medullasin in blood and a method of diagnosing multiple sclerosis using the same. In more detail, it relates to a method of immunologically measuring human medullasin in blood including a step of pretreating the blood sample in order to accurately measure the content of medullasin in the granulocytes in the blood, and to a method of diagnosing multiple sclerosis using the medullasin content in blood.
Medullasin, which is a kind of serine protease, occurs in granulocytes etc. and is thought to widely play important roles in defense mechanism including expression of inflammation, in particular chronic inflammation. The amount of medullasin in granulocytes is increased in advanced stage of a number of chronic inflammatory diseases, and is normalized in remission stage. However, in patients suffering from multiple sclerosis, it is observed that the amount of medullasin is prominently increased a few days before the advanced stage, and is normalized before remission. Multiple sclerosis is characterized by localized demyelinated lesion in white matter of the central nerve system and gliosis. It is a serious chronic inflammatory disease which progresses with repeated remission and aggravation, and in many cases results in death in 10 to 15 years. The cause of multiple sclerosis has still not been clearly identified, but it is thought that this disease is a kind of autoimmune disease in which autoantibodies attack the nerve tissue upon stimulation of the immune system by a virus or a bacterium. Its diagnosis is quite difficult, and is presently carried out by magnetic resonance imaging (MRI) or the like. However, a method such as MRI requires a very large-scale equipment and high skill in the measuring operation and are costly. Furthermore, a method of testing bone marrow liquid has the problem of inflicting much pain on the patient. In light of these circumstances, a simple diagonosis method by which diagnosis of the disease, understanding of the state of the disease and asumption of consequence can be carried out is now being developed. As a result, study has been carried out into methods of measuring the activity of medullasin in granulocyte in blood, and together with the development of an immunological measurement method by which it can be measured easily, there has been proposed the possibility of diagnosing multiple sclerosis according to the granulocyte medullasin content in blood.
However, there has been observed the phenomenon that if, when measuring after diluting the blood-sample with an aqueous medium in a method of immnunologically measuring human medullasin, the measurement is made without carrying out a treatment to completely expel the medullasin present in the granulocyte to out of the granulocyte, the reproducibility of the measured values is not good giving rise to variation in the measured values. Accordingly, the development of a method of immunologically measuring the amount of human medullasin in blood with good reproducibility has been desired.
With respect to the question of whether multiple sclerosis can be diagnosed on the basis of the amount of medullasin in the blood, such a judgment requires the inspection of considerable amounts of clinical data. However, up to now, this kind of data has not existed, and furthermore, it has been difficult to make an accurate diagnosis because of the difficulty in obtaining an accurate measured value of medullasin in granulocyte in blood due to the large variation in the measured values. Accordingly, the diagnosis of multiple sclerosis on the basis of the amount of medullasin in the blood was difficult.
Accordingly, the inventors of the present invention have carried out extensive research into solving the above-described problem. As a result, they found that the human medullasin in blood could be accurately measured with good reproducibility by immunologically measuring the human medullasin using anti-human medullasin antibodies after completely breaking up the leukocytes by treatment of the blood sample with an aqueous liquid including a hemolysate or an aqueous liquid having a specific osmotic pressure different to the osmotic pressure of human blood.
Furthermore, with the establishment of said method for accurately measuring human medullasin with good reproducibility, the inventors of the present invention noticed that the size of and changes in the measured content of human medullasin in the blood sample are closely related to the onset of multiple sclerosis and its extent etc. It was on the basis of these findings that the present invention was arrived at.
A first aspect of the present invention relates to a method of immunologically measuring the human medullasin content in blood characterized by comprising the following steps (a) and (b):
(a) a step of breaking up the leukocytes in a blood sample by contacting said blood sample with the following aqueous liquids (i) or (ii) or an aqueous liquid mixture of (i) and (ii)
(i) an aqueous liquid having an osmotic pressure of 250 mOsm/kg. H2O or less or an aqueous liquid having an osmotic pressure of 310 mOsm/kg.H2O or more;
(ii) an aqueous liquid comprising a hemolysate; and
(b) immunologically measuring the amount of human medullasin released into the blood sample from the leukocytes broken up in step (a) using an anti-human medullasin antibody.
A second aspect of the present invention relates to a method of diagnosing multiple sclerosis characterised by including the following steps (a), (b) and (c);
(a) a step of breaking up the leukocytes in a blood sample by contacting said blood sample with the following aqueous liquids (i) or (ii) or an aqueous liquid mixture of (i) and (ii)
(i) an aqueous liquid having an osmotic pressure of 250 mOsm/kg. H2O or less or an aqueous liquid having an osmotic pressure of 310 mOsm/kg.H2O or more,
(ii) an aqueous liquid comprising a hemolysate;
(b) immunologically measuring the amount of human medullasin released into the blood sample from the leukocytes broken up in step (a) using an anti-human medullasin antibody; and
(c) observing the size of and/or changes in the human medullasin content in the blood measured in step (b).